Unit 7 Flashcards
MOLECULAR DIAGNOSIS OF INFECTIOUS DISEASES
Infectious diseases involve:
○ Viruses
○ Bacteria
○ Fungi
○ detect parasites
- Molecular characterization of microorganisms.
- Development and evaluation of molecular-based laboratory tests of clinical specimens isolated in cultures.
- Comparison of biochemically similar organisms in outbreak situations (known as molecular epidemiology) to ascertain whether the isolates have a common independent source.
APPLICATIONS OF MOLECULAR TECHNOLOGY
APPLICATIONS OF MOLECULAR TECHNOLOGY
- molecular characterization
- for molecular based laboratory tests
- molecular epidemiology
Comparison of biochemically similar organisms in outbreak situations is known as?
molecular epidemiology
Example of molecular epidemiology
SARSCOV (alpha, beta, delta, and omicron strains), slightly different genomes
Analyte for molecular testing used in PCR, RT-PCR, LAMP etc.
genome
mRNA transcript (basis for gene expression)
Transcriptome
proteins of an organism (this is not targeted by molecular tests as much as genome)
Proteum
Proteum is used in which detection method?
MALDI-TOF (Laser desorption targets protein»ionizes»charged)
Pure culture/colony is used in MALDI-TOF
TRUE (ie proteum)
- Time-consuming to isolate organisms
- Hazardous with which to work in the clinical laboratory
- Reliable laboratory tests were lacking
- Organisms that are received in clinical laboratories in high volumes
- Genes that confer resistance to antimicrobial agents
- Characterization of DNA, RNA, and protein to find and identify new organisms and to further characterize or classify known organisms.
COMMON SPECIFIC TARGETS
Hazardous with which to work in the clinical
laboratory
systemic fungi (histoplasma/coccidioides); dangerous to grow in the lab kaya mas safer if molecularly tested
Time-consuming to isolate organisms
TB = takes 8 weeks to be isolated
Reliable laboratory tests were lacking
Hepatitis C
■ Culturing viruses in laboratories is
impractical and costly
■ Can be detected through genomic
testing, protein testing
(antigen/antibody testing)
Organisms that are received in clinical laboratories in high volumes
Streptococcus pyogenes, Chlamydia trachomatis, neisseria gonorrhoeae (last
two often undergo PCR in the Philippines)
ginagamit for transport and collection of swab sample dahil mas maliit yung swab nya; less painful
Stuart transport medium
Genes that confer resistance to antimicrobial agents
Methicillin-, Oxacillin-, Vancomycin-resistant
VRSA Genes
Van A, Van B, Van C, enterococcus
resistance to vancomycin
MRSA Gene
mecA and mecC
■ Staphylococcus
■ Klebsiella
■ Acinetobacter
■ Pseudomonas
■ Enterococcus
1. E. faecalis
2. E. faecium
S.K.A.P.E = To watch out organisms in
cultures; “super bombs”; drug resistant
resistance to rifampicin gene
rpoB (RNA polymerase B gene)
- OR specimens = kapag nilalagyan ng formalin, kasi di mo maeextract yung DNA
- Lysed na yung cells
- Highly fragmented na yung nucleic acids
Rejected samples
Characterization of DNA, RNA, and protein to find and identify new organisms and to further
characterize or classify known organisms.
influenza virus and Sars-CoV
- Viability is not as critical for most molecular testing
- Quality of nucleic acids may be compromised if the specimen is improperly handled
- DNA and especially RNA will be damaged in lysed or nonviable cells
- Avoid contamination that could yield false-positive results
SPECIMEN COLLECTION
only formalinized tissue we use for DNA or RNA extraction
Formalin fixed paraffin embedded tissue
pseudoperoxidase activity in sputum with blood affects downstream application of PCR
TRUE
DNA and especially RNA will be damaged in lysed or nonviable cells because of possible release of nucleases
TRUE
component of sputum with blood that inhibits taq polymerase in PCR
Hemoglobin
PCR IS VERY POWERFUL, but it can saturate the process if very small amount of DNA is used
TRUE
LLOD - Lower limit of detection
amount of DNA used; low LLOD is good! mas sensitive
Principle: Real-time monitoring of PCR progression was performed using fluorescence signal accumulation
Quantitative PCR
Principle: Isothermal Amplification was performed using four specific primers in the presence of Bacillus
Loop-mediated Isothermal Amplification
Principle: Amplification reactions were performed for individual nucleic acid molecules in a separate space
Digital PCR
Principle: The different melting temperatures of mononucleotides lead to different melting curves
High-resolution Melting
Applicability: Routine quantitative detrection of common pathogens such as novel coronavirus, HBV, human papillomavirus, and others in the laboratory
Quantitative PCR
Applicability: Absolute quantification of low-content pathogens such as HBV, HIV, and MTB, etc.
Digital PCR
Applicability: Genotyping of pathogens such as Escherichia coli, Staphylococcus aureus, adenoviruses, etc.
High-resolution Melting
Advantage: Absolute quantification, low sample requirement, and high tolerability
Digital PCR
Applicability: Primary field screening of pathogens such as MTB and Plasmodium.
Loop-mediated Isothermal Amplification
Advantage: Good specificity, high sensitivity, high degree of automation, and low cost.
Quantitative PCR
Advantage: Rapid, high throughput, short time, and low cost
High-resolution Melting
Limitations: Numerous interference factors, time-consuming, instruments are expensive
Quantitative PCR
Limitations: High cost, limited throughput, complex operation
Digital PCR
Advantage: Low instrument requirements, fast reaction speed, low cost, and high sample
Loop-mediated Isothermal Amplification
Limitations: Complex primer design, non-specific amplification, low throughput
Loop-mediated Isothermal Amplification
Limitations: High requirement for uniformity of temperature and primer design
High-resolution Melting
False-positives: Nucleic acid contamination, non-specific amplification
False-negatives: Inhibitors, enzyme inactivation, too little template
Quantitative PCR
False-positive: Nucleic acid contamination
Digital PCR
False-positive: Non-specific amplification
High-resolution Melting
False-positive: Cross-contamination
Loop-mediated Isothermal Amplification
Best dye?
HRM
HRM > Evagreen > SyBr Green 1
TRUE
- Negative derivative in fluorescence and temperature, dalawang peaks ang nakita, edi HRM daw siya
- Means 2 targets yung naamplify mo
- If white peak = naamplify and may nonspecific amplification
TRUE
Polycythemia vera gene
JAK2 (V671E to V617F)
PCR (RT,qRT etc)
DNA Sequencing
Pulse Gel
Electrophoresis
MALDI TOF (mass spectrometry principle)
Molecular methods
- PCR-based amplification.
- can overcome low sensitivity to improve the specificity of conventional phenotypic tests.
- provide early diagnosis for proper treatment with potential reduction in mortality and morbidity.
- all PCR assays have been designed to detect a low load of a given microorganism among a huge number of human cells.
NUCLEIC ACID AMPLIFICATION TECHNIQUES
for MTB/RIF, MRSA, HBV, HIV, HCV, CT/NG (Chlamydia and Neisseria), HPV, SARS-CoV-2, C. difficile, etc.
Cepheid GeneXpert® Systems
Most significant sexually transmitted HPV is 16 and 18
TRUE
<100 CFU/mL = cannot be detected
TRUE
for respiratory, gastrointestinal and cerebrospinal organisms
BIOFIRE® FILMARRAY® SYSTEMS
for HIV, HBV and HCV
Roche COBAS® TaqMan® viral loading systems
Detects through electrophoresis
Seegene AnyplexII HPV HR Detection
- Conserved regions are ideal primer targets for amplification from all bacterial species.
- Measuring the degree of divergence observed within the 16S rRNA molecule.
- It contains well contained regions and variable regions
- The signature sequence obtained is compared to a database containing sequences of known microorganisms.
- The number of similar nucleotide bases between sequences is used to calculate the percent identity and ascertain the identification of the microorganism.
DIAGNOSIS WITH SEQUENCING
- The rRNA genes and their intergenic regions found in bacteria are commonly used for prokaryotic phylogenetic studies.
- The small-subunit rRNA molecule is a fragment with a sedimentation coefficient of 16S and is encoded by a roughly 1542-bp gene.
- 16S is also used for community analysis = gold standard
TRUE FOR DIAGNOSIS WITH SEQUENCING
(beta subunit of RNA polymerase)
rpoB
(manganese-dependent superoxide dismutase)
sodA
(elongation factor Tu)
tuf
- rpoB (beta subunit of RNA polymerase)
- sodA (manganese-dependent superoxide dismutase)
- gyrA or gyrB
- tuf (elongation factor Tu)
- recA
- secA
- Heat shock proteins.
Other DNA targets have been used to better separate closed related species include:
Similar to 16S rRNA gene, these alternative DNA targets have conserved regions flanking variable regions that can be used to differentiate closely related bacterial species.
TRUE
● Bartonellae Species
● Borrelia Species
● Chlamydia trachomatis
● Clostridium difficile
● Mycoplasma pneumoniae
● Rickettsia Species
● Staphylococcus aureus
● Streptococcus pneumonia
● Streptococcus pyogenes
● Streptococcus agalactiae
● ^^Staph and Strep hindi masyado common
DETECTION OF BACTERIAL PATHOGENS
COMMONLY USED PCR TARGETS FOR DETECTION OF Bartonella spp.
- Molecular Characterization of Isolates from Cultures
- Direct Molecular Detection
- mtRNA ssrA
- 16S-23S Internal transcribed spacer (ITS)
- β-subunit of RNA polymerase rpoB
Direct Molecular Detection via real-time PCR
- Citrate synthase gltA
- β-subunit of RNA polymerase rpoB
- 16S-23S Internal transcribed spacer (ITS)
- 16S ribosomal RNA gene 16S
- Riboflavin synthase ribC
- NADH dehydrogenase gamma subunit nuoG
- Cell division protein ftsZ
- Heat shock protein groEL
Molecular Characterization of Isolates from Cultures via conventional PCR
NADH - conventional PCR/RT-qPCR
Sample in MALDI-TOF _______ while in PCR it is ________
pure colony; raw sample
● PCR provides a valuable diagnostic approach in
acutely ill patients.
○ This overcomes the poor sensitivity of
microscopy and can be used either to
diagnose relapsing fever borreliosis or to
further characterize the infecting spirochete.
● Recently applied techniques include NGS and proteomic approaches.
● Not commonly assayed in the PH
BORRELIA SPECIES
Molecular genotyping of ompA can be performed by using restriction fragment length polymorphism on PCR products from culture isolates, but also directly from clinical specimens.
CHLAMYDIA TRACHOMATIS
The nine polymorphic membrane protein genes, pmpA to pmpI, can be used for typing but the discriminating capacity is limited.
CHLAMYDIA TRACHOMATIS
The highly conserved genome of C. trachomatis can be used for multilocus target systems.
TRUE
Analysis of variable numbers of tandem repeats in three loci combined with ompA sequencing can bring a significantly higher diversity index than by using ompA alone.
TRUE
real-time PCRs targeting the ______ toxin genes: tcdA, tcdB and tcdC117.
CLOSTRIDIUM DIFFICILE
Multiplex PCR for the detection of tcdA, tcdB, and the binary toxin (cdtA/cdtB) gene
○ one-step, rapid, and specific screening
method
○ This toxin gene profiling can allow an
evaluation of the pathogenic potential of ____
CLOSTRIDIUM DIFFICILE
Isothermal helicase-dependent amplification for the detection of toxigenic ______
CLOSTRIDIUM DIFFICILE
Development of pseudomembranous colitis
CLOSTRIDIUM DIFFICILE
Both conventional and real-time NAATs have been used to detect ________ RNA.
- monoplex and multiplex format
MYCOPLASMA PNEUMONIAE
LAMP assay also has been applied to detect M.
pneumoniae in clinical specimens using P1 sequences for primers in direct comparison to real-time PCR.
TRUE
A multiplex real-time PCR assay can be used to detect point mutations in all three positions of the 23S rRNA gene (___, ____, and ____) that are related to the macrolide resistance on all clinical samples that are positive for M. pneumoniae in the repMp1 real-time PCR assay.
2063, 2064, 2617
Specimen:
- Whole Blood/
- Buffy coat
- Serum/ Plasma
- Skin/eschar biopsy and autopsy organ tissue
- Eschar Swab
- Formalin-fixed tissue; paraffin-embedded tissue
RICKETTSIA SPECIES
Preservation and Transportation for Rickettsia is -20°C; >24hrs, EXCEPT:
Eschar Swab: -2-8°C; >24hrs
Formalin-fixed: Room Temperature
a leading cause of bone and joint infections and one of the most common causative pathogens of bacterial pneumonia in children.
STAPHYLOCOCCUS AUREUS
____ and ___ are the main genes responsible for the resistance of MRSA to most of the β-lactam antibiotics.
mecA and mecC
PCR is considered to be the best molecular diagnostic tool for MRSA detection.
TRUE
Duplex PCR assay detecting mecA and ____ genes can be very useful.
femB/nuc
A triplex PCR targeting 16S rRNA, mecA, and nuc genes can reach 98% accuracy within 6h of visible growth detection.
TRUE
Pneumococcal surface adhesion A (psaA), pneumolysin (ply), penicillin-binding protein (PBP), and autolysin (lytA)
STREPTOCOCCUS PNEUMONIAE
Real-time PCR: Analyzing sputum through real-time PCR with the targets at lytA.
STREPTOCOCCUS PNEUMONIAE
- GASDirect test identifies specific rRNA sequences of S. pyogenes in pharyngeal specimens by a single-stranded chemiluminescent nucleic acid probe
- Can be applied for primary testing, has been used as a backup test to negative antigen tests, and is suitable for batch screening of throat cultures.
STREPTOCOCCUS PYOGENES
Specimens used:
- vaginal-rectal swabs, amniotic fluid, neonatal
screening swabs, blood, breast milk, urine,
and serum.
STREPTOCOCCUS AGALACTIAE
- LAMP Method can amplify target nucleotide sequences at isothermal conditions (usually 60-65°C) within 90 mins by using four or six primers.
- Amplification can be detected by macroscopic observation of turbidity or color change of a fluorescent dye.
STREPTOCOCCUS AGALACTIAE
Developed successfully by using probes targeting cfb gene.
STREPTOCOCCUS AGALACTIAE
Universal 16S PCR also has been used to identify GBS (Group B Streptococcus) from blood, bone, and joint infections.
TRUE
Targets for GBS-PCR include:
sip gene - codes for the Sip immunogenic protein.
cfb gene - codes for the Christie-Atkins-Munch-Petersen factor.
scpB gene - codes for the C5a peptidase
ptsI gene - codes for phosphotransferase.
TRUE
codes for the Sip immunogenic protein
Sip gene
codes for the Christie-Atkins-Munch-Petersen factor
cfb gene
codes for the C5a peptidase
scpB gene
codes for phosphotransferase.
ptsI gene
For differentiation between strains of the same fungal species, multilocus sequence typing of housekeeping genes can be used.
TRUE
PCR-based DNA amplification or ______
isothermal amplification
Genus or species-specific primers and probes methods:
- Southern blotting, slot blotting, PCR-enzyme immunoassay microarray, nested PCR, PCR-restriction fragment length polymorphism, and microsatellites
TRUE
Nested PCR can be used to detect Aspergillus and Candida.
TRUE
Tissue is processed frequently for histopathology with FFPE tissue, which hampers the molecular identification workflow because of technical issues.
TRUE
Fresh frozen tissue gives a better yield than FFPE tissue in qPCR detection.
TRUE
Target Gene: Actin
Aspergillus
Target Gene: β-Tubulin
Phaeacremionium
Aspergillus
Pseudallescheria
(PAP)
Target Gene: Calmodulin
Aspergillus
Pseudallescheria
(AP)
Target Gene: Chitin synthase 2
Lacazia loboi
Target Gene: Cytochrome b
Aspergillus
Trichosporon
Rhodotorula
(ATR)
Target Gene: D1-D2
Most fungi
Target Gene: Elongation factor 1a
Fusarium species
Target Gene: ITS
Medically significant yeasts
Fungi
Target Gene: 26S
Medically relevant Fusarium and Scedosporium
For differentiation between strains of the same fungal species, multilocus sequence typing of house-keeping genes should be used.
TRUE
For direct sequencing, the ___ and ___ fragments can be used for PCR amplification.
26S and 28S
_______ is highly sensitive and specific for the detection of Aspergillus DNA or RNA.
NAAT
Real-time PCR can be used to quantify fungal burden
TRUE
Isothermal amplification techniques, such as nucleic acid sequence-based amplification (NASBA), detect Aspergillus nucleic acids
TRUE
In situ hybridication (ISH) probes targeting fungal rDNA have been used for the detection of Aspergillus in fresh and FFPE tissues without requiring nucleic acid extraction or amplification. This enables?
enables direct visualization of organisms with the use of labeled probes that bind to complementary fungal sequences.
rDNA gene cluster: most common target of assays designed for direct detection in clinical specimens because of the presence of highly conserved (18S rDNa and 28S rDNA) and variable (ITS and D1/D2) regions.
TRUE
Genus level assay designs often target ____
18S
A. fumigatus-specific assays target ITS-1, mitochondrial DNA, alkaline protease, or Aspergillus collagen-like (acl) genes.
TRUE
A novel, simple real-time qPCR use a probe as a primer to initiate PCR amplification under the high fidelity (HF) DNA polymerase and the emitted fluorescence during the amplification can be monitored during a real-time PCR amplification.
DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION
Tolerance to mismatches between primer/probe and target and the one primer-one HF man probe system simplifies the method, improves the sensitivity and accuracy.
DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION
_______________-mediated qPCR method is a simple, sensitive, and promising approach for the diagnostics for viral infectious diseases.
HF DNA polymerase-mediated qPCR method
- Some HPV types have the potential to cause lesions that progress to cancer.
- The highest risk for cancer development occurs as a result of prolonged persistent infection over many years.
TRUE
The majority of molecular diagnoses are designed to detect nucleic acids of the 12 IARC HPV group 1 carcinogens
HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and 59
Multiplex real-time PCR and nucleic acid hybridization with four different fluorescent reporter probes can be used to concurrently detect the L1 gene of HPV16 and HPV18 as individual reactions and HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68 as a pooled result.
TRUE
L1 gene of HPV16 and HPV18 as ____ reactions
individual
HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68 as _____ result
pooled result
Detection of HPV E6/E7 oncogene mRNA in cervical cells is an alternative to detection of HPV DNA.
TRUE
HPV tests based on nucleic acid sequence-based amplification (NASBA) can detect very little expression of E6/E7 mRNA for transient infection.
TRUE
Persistent infections = overexpression of _____
E6/E7 mRNA
__ refers to dominant promoter
P97
All PVs are similar in their genetic organization and appearance under electron microscope.
TRUE
URR
Upstream Regulatory Region
Real-time PCR is one of the latest techniques frequently used to diagnose viral hepatitis.
TRUE
PCR-based assays, coupled with oligonucleotide microarray technology, allow for the simultaneous detection and genotyping of several viruses, including blood borne pathogens, respiratory viruses, and adenoviruses.
TRUE
Multiplex qPCR assays use the most conserved region representing all the strains/variants for detection of the hepatitis viral genome in body fluid.
TRUE
Multiplex qPCR has been employed successfully for the simultaneous detection of hepatitis virus A, B, C, D, and E and genotyping of their strains.
TRUE
A single-step multiplex qPCR assay allows for an early diagnosis and timely treatment of patients with viral hepatitis.
TRUE
HAV Virus, HCV, HGV
5’ UTR
HBV
S and X gene
HDV
Ribozyme-1
HEV
ORF2 and ORF3
Segment __ is complementary to the HBV DNA plus strand from nt 1615 to nt 1604
A
Segment __ is consensual to the HIV long terminal repeat (LTR) region, which differs from the HBV DNA sequence
B
To reduce rcDNA contamination: a chimeric sequence composed of two segments to increase specificity: Segment A and Segment B
TRUE
Can effectively distinguish cccDNA from other HBV DNa
TRUE
______ is regarded as the best test for quantitative cccDNA detection.
Southern Blotting
The higher the mass, the longer the time of flight of the ion.
TRUE
A peptide is placed on a matrix, which causes the peptide to form crystals
Laser pulse is used to excite the chemical matrix, creating a microplasma that transfers the energy to protein molecules in the sample, ionizing them and ejecting them into the gas phase.
Protein molecules that have packed up a single proton can be selected by the MS for mass analysis.
TRUE
The general procedure for the identification of bacteria and yeast by MALDI-TOF MS using the intact-cell method
TRUE
● 16S rRNA gene
● 16S-23S ITS (locus)
● gltA (citrate synthase)
● rpoB (β-subunit of RNA polymerase)
● ribC (riboflavin synthase)
● nuoG (NADH dehydrogenase gamma subunit)
● ftsZ (cell division protein)
● groEL (heat shock protein)
● ssrA (transfer-messengerRNA)
Bartonellae spp
● ompA
● pmpA - pmpl
Chlamydia trachomatis
● 16s rRNA gene (commonly targeted;
broad-range)
Borrelia spp
● tcdA
● tcdB
● tcdC117
● Binary toxin (cdtA/cdtB) gene
Clostridium difficile
● P1 sequences
● 23S rRNA gene (positions 2063, 2064 and 2617)
● repMp1
Mycoplasma pneumoniae
● 16s rRNA gene (commonly targeted; broad-range)
● 17kDa antigen gene
Rickettsia spp
● mecC gene
● 16s rRNA
● mecA gene
● Nuc genes
^^last 3 in triplex PXR = 98% accurate
Staphylococcus aureus
● psaA (Pneumococcal surface adhesion A)
● ply (pneumolysin)
● PBP (penicillin-binding protein)
● lytA (autolysin)
○ ^^Sputum real time-PCR
Streptococcus pneumoniae
● 16s rRNA gene (commonly targeted; broad-range)
● emm gene
Streptococcus pyogenes
● 16s rRNA gene (commonly targeted; broad-range)
● sip gene (Sip immunogenic protein)
● cfb gene (Christie-Atkins-Munch-Petersen factor)
● scpB gene (C5a peptidase)
● ptsI gene (phosphotransferase)
Streptococcus agalactiae
● Actin
● rDNA gene cluster
● 18S
● ITS-1
● Mitochondrial DNA
● Alkaline protease
● acl genes
^^ last 3 (A. fumigatus-specific)
Aspergillus
● L1 gene
● E6/E7 oncogenes
Human Papillomavirus
● Conserved regions of the viral genome (see
table below)
● cccDNA (for HBV)
Hepatitis virus
